Fluorescein is a commonly used labeling material for protein, in particular in the context of immunoassays. Immunoglobulins can be readily made detectable by conjugation to fluorescein.
Typically, this conjugation is effected by reacting commercially available fluorescein isothiocyanate (FITC) with the compound to be labeled. This results in a thiourea linkage involving the isothiocyanate of FITC and free amino groups on the protein. The resulting thiourea-linked fluorescein has diminished fluorescence as compared to fluorescein itself. The corresponding urea derivatives, which would be the products of the analogous reaction of the amino groups of the proteins with fluorescein isocyanate, have higher fluorescence intensities, but have not been used routinely because the starting material, fluorescein isocyanate, is unstable and thus must be freshly prepared before use. Nairn, R. C., "Fluorescent Protein Tracing", 4th ed. (1976), Churchill-Livingston, NY, p. 17.
It has now been shown that replacing the thioureido group with a thioacetamido, or, preferably, acetamido, subunit enhances the ability of the resulting compound to fluoresce. Accordingly, compounds have been designed which contain the configuration --NHCSCR.sub.2 --, or preferably --NHCOCR.sub.2 --, in the region immediately adjacent the fluorescein nucleus.
Others have used fluorescein linkages not derived from FITC. For example, Wang, C. H. J., et al, Canadian patent 1,178,269, discloses a method for determining ligands in a sample using labeled compounds wherein a carbonyl group rather than the isothiocyanate residue is directly attached to the fluorescein moiety. Thus, these ligands are directly bound to 4- or 5-carboxyfluorescein. They are formed in a procedure whereby the carboxy group has been activated to permit binding to the desired ligand. According to the reference, these compounds were used to prepare "tracers" for a fluorescence polarization assay, and there is no discussion of the fluorescence efficiency of the conjugates. The disclosure in this reference also refers to the use of carboxyfluorescein by others in forms not conjugated to biological molecules (Chen, R. F., Anal Let (1977) 10: 787).
A paper by Franzen, J. S., et al, Biochemistry (1980) 19: 6080-6089, refers to the reaction of 5-iodoacetamidofluorescein (and the corresponding eosin) with an enzyme previously labeled with 5-[(iodoacetamido)ethyl)amino]naphthalene-1-sulfonic acid. No special fluorescence intensity properties of these compounds are reported; the donor/receptor relationship of the fluorophores was used to study the locations of the catalytic sites.
It should be noted that compounds derived from fluorescein that contain a halide immediately adjacent the methylene group in the --NHCOCH.sub.2 -- side chains of the invention are commercially available; one such compound, mentioned above, is 5-iodoacetamidofluorescein. In addition, 5-acetamidofluorescein is known to fluoresce at an intensity comparable to that of fluorescein per se. Derivatives of oxidized sugars containing the amide linkage also fluoresce at high intensity levels. These labeled sugars can be then tracked metabolically (Ogano, H., et al, Carbohydrate Res (1982) 105: 69-85).
Also, derivatives of steroids using an acetamide type linkage of fluorescein with steroids have been prepared. Four such compounds have CA registry numbers 92264-85-2, 92264-86-3, 93800-97-6 and 61925-79-9; they are evidently prepared by condensation of a steroid-linked carboxyl group with fluoresceinamine. A derivative of the amino derivative of eosin with a fatty acid is also described (Blatt, E., et al, Biochim Biophys Acta (1986) 857: 85-94).